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FLP-containing pBBR1MCS-2 recombinant plasmid and method for modifying zymomonas mobilis genome DNA

A technology of Zymomonas mobilis and recombinant plasmids, which is applied in the field of microbial genetic engineering, can solve the problems of inefficiency, difficulty in satisfying the application research of Zymomonas mobilis molecular biology transformation, singleness, etc.

Inactive Publication Date: 2011-02-16
TIANJIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] To sum up, the current methods for the modification of Zymomonas mobilis genome DNA are still relatively simple and inefficient, and it is difficult to meet the needs of Zymomonas mobilis

Method used

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  • FLP-containing pBBR1MCS-2 recombinant plasmid and method for modifying zymomonas mobilis genome DNA
  • FLP-containing pBBR1MCS-2 recombinant plasmid and method for modifying zymomonas mobilis genome DNA
  • FLP-containing pBBR1MCS-2 recombinant plasmid and method for modifying zymomonas mobilis genome DNA

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Experimental program
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Effect test

Embodiment 1

[0033] Construction of embodiment 1pBBR1MCS-2-Pgap-FLP

[0034] For a schematic diagram of this build see figure 1 , see Table 1 for the primers P1 to P4 involved.

[0035] Table 1. Primers used for construction and identification of Zymomonas mobilis engineering strains in the present invention

[0036]

[0037] Using the genomic DNA of Zymomonas mobilis sequencing strain ZM4 as a template, primers P1 and P2 were designed according to the published ZM4 genome sequence AE008692, and a 347bp ZM4-containing glyceraldehyde 3-phosphate dehydrogenase gene gap promoter was amplified The product of the region is named PCR1;

[0038] Using the pCP20 plasmid as a template, a 1474bp product containing the Saccharomyces cerevisiae site-specific recombinase gene FLP was amplified with P3 and P4, which was named PCR2;

[0039] Then PCR1 and PCR2 were used as templates, and P1 and P4 were used as primers to amplify to obtain a 1801bp product, which was named PCR3. PCR3 contained ClaI ...

Embodiment 2

[0042] Embodiment 2: Construction of ZM4 (ldh::FRT) strain ZM4 (ldh::FRT) modified by inactivation of the lactate dehydrogenase gene ldh of Zymomonas mobilis ZM4 genome

[0043] 1. At site 1 (lactate dehydrogenase gene ldh) of Zymomonas mobilis ZM4 genomic DNA, integrate a DNA fragment (ie FRT-cml-FRT) containing two FRT sites in the same direction, said DNA fragment The selection marker gene chloramphenicol gene cml is contained between the two FRT sites of , and the strain ZM4 (ldh::FRT-cml-FRT) modified at site 1 is obtained. For the construction of this bacterial strain, see the literature (Zou Shaolan, Hong Jiefang , Ma Yuanyuan et al. Construction of targeted integration plasmids for Zymomonas mobilis, Journal of Nankai University (Natural Science Edition), 2009, 42(6): 42-47);

[0044] 2. Introduce pBBR1MCS-2-Pgap-FLP into the strain ZM4(ldh::FRT-cml-FRT) constructed in step 1. by electroporation, and screen out the deletion of the two FRT sites due to FLP expression. ...

Embodiment 3

[0055] Example 3: Construction of ZM4 (ldh::FRT) (gfo::FRT-tktA talB) modified by inactivation of the glucose-fructose oxidoreductase gene gfo in Zymomonas mobilis ZM4 (ldh::FRT) genome

[0056]1. At site 2 (glucose-fructose oxidoreductase gene gfo) of Zymomonas mobilis ZM4 (ldh::FRT) genomic DNA, a DNA fragment (ie FRT-cml- FRT-tktAtalB), the DNA fragment contains the selectable marker gene cml between the two FRT sites, resulting in modified ZM4 at site 2 (ldh::FRT) (gfo::FRT-cml-FRT-tktA- talB) strain

[0057] (1) Construction of integrated plasmid pUC19-gfoR-FRT-cml-FRT-tktA-talB-gfoL

[0058] See the whole construction process image 3 . The cloning primers P7 to P10 and identification primers P11 to P12 used are shown in Table 1.

[0059] Primers P7 to P10 were designed according to the published ZM4 whole genome sequence AE008692. Using the genomic DNA of the Zymomonas mobilis sequencing strain ZM4 as a template, PCR was amplified with P7 and P8 to obtain PCR1, whi...

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Abstract

The invention discloses an FLP-containing pBBR1MCS-2 recombinant plasmid and a method for modifying a zymomonas mobilis genome DNA. The plasmid is constructed by the step of: inserting a segment Pgap-FLP into pBBR1MCS-2 through C1aI and XbaI sites to obtain pBBR1MCS-2-Pgap-FLP, wherein the Pgap-FLP is a nucleotide sequence shown in SEQ ID NO:1 in a sequence table. The plasmid can be conveniently transferred in an electroporation manner to enter zymomonas mobilis, and can be conveniently removed. By combining with an FRT-FLP site specificity recombinant system from saccharomyces cerevisiae, the invention provides a rapid, efficient, convenient and stable method for modifying zymomonas mobilis genome DNA. The invention provides a method for modifying DNAs at multiple sites of the zymomonas mobilis genome by circularly utilizing a selective marker gene.

Description

technical field [0001] The invention belongs to the field of microbial genetic engineering. Specifically, it relates to a FLP-containing pBBR1MCS-2 recombinant plasmid and a method for modifying Zymomonas mobilis genome DNA. Background technique [0002] With the implementation and completion of various genome sequencing projects including the Human Genome Project, genome function research based on sequence information has immediately become another major topic in the field of life sciences. Compared with sequencing engineering, genome functional research is more complex and difficult. For a specific gene, in order to fully understand its biological function, it is often necessary to clone, delete, and mutate the genomic DNA containing the complete information of the gene, so as to lay the foundation for subsequent expression and function research. Zymomonas mobilis, as a microorganism with great industrial application value and two typical strains have been sequenced, als...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/63C12R1/01
Inventor 邹少兰洪解放马媛媛张鲲井欣张敏华
Owner TIANJIN UNIV
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